Substantially pure solid form of the enol tautomer of 3-indolypyruvic acid for use in the treatmetn of central nervous system disturbances

ABSTRACT

The enol tautomer of 3-indolylpyruvic acid in stable and pure form, as well as alkali and alkali-earth salts thereof are produced by synthesis process. The enol tautomer comprises only the geometric isomer (Z) and it is the only one tautomeric form having relevant pharmacological effects. The compounds obtained exhibited relevant biochemical and pharmacological properties in pathological situations like anxiety, depression, behaviour disturbances, neuronal degenerations, etc.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention refers to the enol tautomer of 3-indolylpyruvicacid (briefly IPA) obtained in an essentially keto tautomer-free form,suitable for the employ in pharmaceutical industry treatment steps, aswell as to salts thereof, particularly to alkali and of alkali-earthmetal salts.

The invention further refers to a process for the production of theabove cited enol tautomer and of the salts thereof in solid form,preferably of essentially crystal structure, as well as their activityas therapeutically active agents, specifically in the treatment ofcentral nervous system disturbances.

2. Description of the Prior Art

Keto-enol tautomerism is a well-known chemical phenomenon, definable asthe entirely reversible migration of a Hydrogen atom from a Carbon inalpha, adjacent to a carbonyl group, with a subsequent double bondshift. The forms thus generated have distinguishable chemico-physicalfeatures, and are respectively defined as keto and enol ‘tautomers’. Ina solution, all keto species, alpha-ketoacids included, are subjected todynamic transformation of a tautomer into the other until attaining anequilibrium situation, whose extent depends on factors such as thechemical species considered, the solvents, the pH, the temperature, etc.However, tautomers, being intrinsically transformable into each otherwith the utmost ease, are not usually treated as chemical species perse, i.e. capable of giving rise to clearly distinct biochemicalreactions, being instead generally assessed for their end effects insolutions, as if being a single compound.

In the case of stereoisomers, a spontaneous conversion of the isomericforms into each other is normally impossible to occur. In particular, ifa first of the forms is the therapeutically active one, the otherenantiomer is considered as an impurity, which can be eliminated byconventional purification methods.

On the contrary, in the case of the tautomers, these convert into eachother spontaneously even under mild conditions, until they arrive at abalance situation. In fact, the tautomers are a dynamic system in acontinuous and macroscopic modification depending on the experimentaland preservation conditions. Consequently, while it is still possible tostate by conjecture that only one of the tautomeric forms is thattherapeutically active, the other tautomer can not simply be consideredas an impurity to be eliminated, but as a species spontaneously takingpart to the structural balance. By this spontaneous mechanism of action,the “concentration” of the therapeutically active species will always bemade uncertain.

3-indolylpyruvic acid (3-indolylpropionic-α-oxo acid) is analpha-ketoacid widespread in the vegetable and animal world. It beingthe transamination product of tryptophan, an amino acid of the naturalseries, essential in the diet of several animals and found in mostproteins, the former is certainly present in all living cells, thoughits quantitative determination has always entailed several technicalproblems.

For instance, for several decades now it has been known that3-indoleacetic acid, acting as growth factor in plants, directly derivesfrom the decarboxylation of 3-indolylpyruvic acid, which in turn isobtained by transamination from tryptophan. However, the studiesconducted to quantify the extent of these transformations had seriousdifficulties: firstly, the various methods used to synthesize3-indolylpyruvic acid led to multicolored compounds, whosechemico-physical behaviour proved erratic and depending on the specificsynthesis method used; secondly, the presence of the keto-enoltautomerism hindered the clear identification of the chemical speciesoriginated by tryptophan transamination. Several decades ago, in anattempt to understand the features of the keto-enol tautomerism of3-indolylpyruvic acid many basic works were conducted, which yieldedhighly interesting results, above all considering the limitations in theinstrumentation available at that time.

E.g., J. A. Bentley et al. (Biochemical J. 64, 44-49, 1956) demonstratedthat freshly synthesized 3-indolylpyruvic acid behaves in a whollydifferent manner in paper chromatography, depending on the systems usedas eluent: a solution consisting of isopropanol, ammonia and watercompletely destroys the alpha-ketoacid, whereas the acetic acid-watermixture allows to obtain two distinct blots. Always using paperchromatography, Schwarz and Bitancourt (Biochemical J. 75, 182-7, 1960)demonstrated 3-indolylpyruvic acid to be an extremely unstable substancethat cannot be adequately singled out in chromatograms, either becauseit is completely destroyed, or because the solvents used do not separateit from its decomposition products: adopting proper care and abidimensional chromatogram, two distinct products could be obtained,deemed to be the keto and the enol tautomers, plus several otherunidentified decomposition products. In the same work, it is reportedthat probably the ketoacid precipitates in a mainly enol form, yet in asolution at pH 8.0 it was largely converted to the keto form in 20minutes. Schwarz (Archives Biochem. Biophys. 92, 168-75, 1961) describedthe separation by paper chromatography of the two tautomers of3-indolylpyruvic acid, followed by UV spectrum identification: thetypical spectrum of the enol tautomer gradually converted, in over 2hours, to that of the keto tautomer, whereas the spectrum of the ketoform rapidly converted to that of the enol form, in the presence ofboric acid and of tautomerase (an enzyme facilitating interconversionbetween the two forms). In two accurate works, J. M. Kaper et al.described studies on 3-indolylpyruvic acid conducted by paperchromatography (Archives Biochem. Biophys. 103, 469-74, 1963) and ontheir UV spectra (Archives Biochem. Biophys. 103,-475-87, 1963). Theyreported that in ammonia environment the paper chromatography of3-indolylpyruvic acid rapidly leads to a decomposition toward at least 7products of indolic nature, of which only a few were identified. Thework concludes stating that, under the conditions used, a rapidconversion from the enol to the keto form was apparent. However, thereaction subsequently progressed to degradation products, stimulated bythe presence of oxygen, light, ammonia, or even of solvent contaminants.Concerning the spectrophotometric studies, they confirmed that, while3-indolylpyruvic acid in solid form is mostly found as enol, whendissolved it assumes an equilibrium situation that always favours theketo form. Moreover, pH seems to markedly influence the keto-enolequilibrium, since in the acid region (pH 2.9-6.0) the enol form isreasonably stable, whereas in the basic region (pH 7.7-9.5) the spectrumof the keto form rapidly predominates. Lastly, Nazario and Schwarz(Archives Biochem. Biophys. 123, 457-61, 1968) conducted studies on theketo-enol equilibrium of 3-indolylpyruvic acid, using IR spectrometry.They reported that, while the enol form of the ketoacid predominates inthe solid form, in acid environments, in aprotic solvents or inmethanol, the keto form predominates in non-acid aqueous solvents and inthe presence of protophilic solvents.

Therefore, overall, the studies published in the '50s and in the '60slead to conclude that 3-indolylpyruvic acid is a compound characterizedby great instability, precipitating mainly in the enol form. Howeverwhen the solid is dissolved in an aqueous solution, the equilibrium isswiftly shifted to the keto tautomer and in physiologic solution “invitro” the specific pattern of the enol tautomer disappears after a fewminutes. In fact, in aqueous solution at neutral or basic pH IPA rapidlyreaches an equilibrium in which the keto form markedly predominates.Accordingly, the ketoacid tautomers should be consideredindistinguishable in their physiopathological effects on plants andanimals, since, due to the the fixed temperature and pH conditions inthe cell environment, the equilibrium reached in solution between thetautomers of 3-indolylpyruvic acid is always characterized by the markedpredominance of its keto form, and this will determine the metabolicdestiny of the ketoacid.

Therefore, according to the prior art teaching, it was irrelevant tohave a lower or higher proportion of enol tautomer, in that it wasexpected that “in vivo” only the keto tautomer would have been present.

Technical problem of the invention.

The literature on 3-indolylpyruvic acid reports, already from the '50sand '60s, that the ketoacid, obtained via different synthesis methods inorganic solvents, precipitates mainly in the enol tautomeric form.However, it has been observed that the presence of impurities(consisting of both the keto tautomer and the other compounds obtainedas secondary products in synthesis processes) represents a criticalfactor in determining the chemico-physical features of the powdersobtained, as well as the metabolic destiny of the molecular species inthe solution, hence outmost care should be exerted in obtaining enolform at the maximum purity grade theoretically attainable.

Moreover, as a result of the present invention and the pharmacologicaltests carried out with the products thereof, it has surprisingly beenfound that the sole enol form of 3-indolylpyruvic acid, thus excludingthe keto form, is accountable for the favourable pharmacological resultslinked to the therapeutic use of 3-indolylpyruvic acid. Hence, thestability of the enol form becomes a critical factor in thepharmaceutical utilization of 3-indolylpyruvic acid.

Furthermore, based on the present invention, it has been found that:

-   -   a) contrary to what occurs “in vitro”, human and animal biologic        fluids treated by the enol tautomeric form show that the enol        tautomer is stable for many hours (likely by action of        tautomerases);    -   b) the keto tautomer form is practically devoid of any        pharmacologic activity, whereas the the enol form shows a wide        pattern of activity useful in therapy,    -   c) the salification of the enol form allows the use of the enol        compound also through an injection route of administration, when        it can not be administered orally.    -   d) further to the practically pure enol form of IPA (enol purity        99,9%), it has been found that the enol form itself as obtained        with the process of the present invention, surprisingly consists        exclusively of the (Z) geometric isomeric form of the enol        tautomer, instead of being a mixture of the two possible        geometric isomer forms (E) and (Z).

Consequently, obtaining the enol tautomer of IPA in the state of highestpurity both of the enolic and the (Z) form of isomers, makes possible toattain foreseeable and reliable therapeutic effects, contrary to thecase in which also the keto tautomer was present from the beginning.

Hence, an object of the present invention is to obtain the enol form of3-indolylpyruvic acid in a physical state allowing a reliablepharmaceutical use on the drug manufacturing stage and a reliabletherapeutic utilization of said drugs. Therefore, the object is that ofobtaining the enol tautomer form in a semicrystalline solid state and ina virtually pure state, essentially free from the keto form of3-indolylpyruvic acid. This means that such virtually pure state isequivalent to a theoretical resolution of the enol form with respect tothe keto-enol tautomerism of 3-indolylpyruvic acid.

The prior art reports several methods for obtaining 3-indolylpyruvicacid: apparently, some methods (e.g those described in U.S. Pat. No.4,551,471 and in U.S. Pat. No. 5,002,963) are not useful to obtainsolely the enol tautomer of 3-indolylpyruvic acid, as there are alsoobtained large amounts of the keto form of 3-indolylpyruvic acid, fromwhich it is not possible to obtain the corresponding high-purity enolform anymore.

Other methods described (es. Hoppe-Seylers z. 109, 259, 1920; Biochem J.64, 44, 1956; Arch. Bioch. Biophys. 103, 469, 1963; J. Biochem. 44, 47,1957; U.S. Pat. No. 5,210,215) allow to obtain more than the 90% of enolform, yet the compounds are often polluted by residues of othersubstances of indolic nature and, above all, by colored substances whosechemical characterization is virtually impossible, and deriving fromfree radical capture phenomena and subsequent polymerization phenomena.

U.S. Pat. No. 4,808,728 to the same Applicant also teaches a process forthe production of 3-indolylpyruvic acid. The product is obtained with ahigh purity grade, yet such process fell short of providing the resultsadvanced by the present invention. Moreover, the product obtained has astructure of flocculent nature, instead of a semicrystalline structure,being therefore quite unsuitable for pharmaceutical processing and ofuncertain stability.

U.S. Pat. No. 4,808,728 describes a synthesis method of preparation ofindolyl pyruvic acid (briefly IPA). The tautomeric features thereof arenot specified in that patent disclosure. The recovered acid has a goodpurity level (absence of polymerization products, other substances ofindole structure and color), and an acceptable chemical recovery yieldof about 50%, with an amorphous structure of the solid precipitateproduct.

The above mentioned method was a useful starting point on the way to thepresent invention. However in the present invention, a plurality ofmodifications to the synthesis procedure of U.S. Pat. No. 4,808,728 havebeen made. A remarkable yield increase in the recovery (higher than 80%)of IPA in its (Z)-enol tautomeric form has been obtained with an enolpurity higher than 99,9% and a recovered solid precipitate ofsemicrystalline structure having an industrially useful shelf life.

Furthermore, it has been surprisingly found that while can exist twopossible geometric isomers of the enol tautomeric form of IPA, which arethe geometric E and Z isomers, exclusively the geometric isomer Z isformed in the synthesis of the present invention.

This result will be evidenced by the data of 1-H and 13-C NMR analysisof the product, which will be shown later in this description.

The results obtained with the present invention can be summarized asfollows:

-   -   a) only one of the two tautomeric structures is formed (the        enolic form) with high yields in chemical conversion,    -   b) the enolic form obtained consists exclusively of the        geometric isomer Z of IPA,    -   c) this Z form is a precipitate having the highest level of        chemical purity,    -   d) the physic state of the precipitate is a semicrystalline        structure, highly homogeneous in structure and color and of easy        workability and recovery.    -   e) the solid precipitate has a high shelf life structural        stability (absence of both a spontaneous conversion of one        tautomer into the other with no formation of degradation        products) when maintained in the normal preservation conditions        of a pharmaceutical raw material.

In connection to the above, it is to point out that geometric isomers(E) and (Z) generally due to the presence of a double bond, as occurringin the enolic IPA structure, can usually have even very differentchemico-physical properties. As an example, one can consider the extremecase of difference between fumaric acid (geometric (E) isomer) andmaleic acid (geometric (Z) isomer).

Therefore the teaching deriving from prior art U.S. Pat. No. 4,408,728was that in the method of production of IPA a spontaneous tautomericbalance would occur between the keto and enol tautomeric forms. The enolform, in turn, would find an a priori unforeseeable balance between thepercentages of the two geometric isomers (E) and (Z) respectively.

As a consequence of the possible differences in the chemico-physicalproperties of the geometric isomers, also the pharmacological propertiesthereof should be expected to be different.

In view of the above, the IPA produced according to the prior art wascomprising a mixture in variable proportions of the two tautomeric formsand of the two geometric isomers. This resulted in a not clearpharmacological profile of IPA as well as in unreliable results of thepharmacologic tests.

In the present invention a IPA is provided consisting of only onetautomeric form never individually insulated in the prior art in astable and pure state. In addition the enolic form as obtained consistsof only one precisely identified geometric isomer form, not known in theprior art as a pure and stable individually resolved enantiomer. Thisproduct is obtained at the highest chemical purity level, and moreoverit has the unexpected property of a shelf life comparable to the usualpharmaceutical raw materials. This now allows for the first time in thepharmacological art to carry out tests on IPA with expectance ofunambiguous and reliable results.

According to the present invention, 3-indolylpyruvic acid in a virtuallypure enol form (≧99% purity, absence of keto form and of polymerizationproducts) is obtained for carrying out the subsequent biochemical andpharmacological tests with certainty of product stability.

A further object of the present invention is a process providing theproduct in a nearly entirely needle-shaped crystal form, anyhow whollysuitable to be employed by the pharmaceutical industry and for thepreparation of capsules, tablets, injection ampoules.

In above mentioned U.S. Pat. No. 4,808,728 (Example 4) high care wastaken to maintain a reaction environment anhydrous as much as possible.In fact the focal point in the transformation of tryptophan into IPA wasthe condensation reaction of the aromatic aldehyde with the aminefunction, the fundamental water removal of water from the adduct and therearrangement thereof from aldo-amine to keto-imine.

In view of the above preliminary assumptions the use clear of “powerful”reactants, such as 1,8-diazabicyclo(5.4.0)-undec-7-ene (briefly DBU) asa dehydrating non-nucleophilic base and anhydrous ZnCl₂ as a complexantagent as well as the operation under controlled temperature and “stiff”reaction times.

Thus, the procedure of the cited prior art is carried out under “forced”conditions and reactants. This brings the consequence of a relativelynarrow window of operating conditions.

On the contrary, the method of the present invention starts from adifferent philosophy based on a better manageable “soft” approach, whichwill be hereinafter disclosed in comparison to the cited U.S. Pat. No.4,808,728. In this discussion, the particular distinguishing featuresare pointed out which have brought to the far superior results obtainedwith the present invention.

A) Use of triethylamine (TEA) in lieu of DBU.

Advantages: reduced heat, better temperature and reaction time control,no need of anhydrous solvents, a weaker base and so reduced sidereactions, lower cost, lower toxicity, more soluble intermediate salts.

Both DBU and TEA are tertiary aliphatic non-nucleophilic bases. Howeverthey have remarkably different basic characteristics (higher for DBU),while they show also a remarkably different volatility (higher for TEA).In view of this in a synthetic procedure development usually DBU ispreferred to TEA, rather than the contrary. With the present inventionit was found that TEA could perform the role of DBU with the advantageof a minor chance of side reactions.

In addition TEA allows an easier and precise temperature control. It wasin fact discovered that an excessive and uncontrolled temperature of thereaction mixture at this procedure step reduced the IPA yield andquality.

B) Use of Zn acetate bihydrate in lieu of anhydrous ZnCl₂.

In the prior art procedure the use of anhydrous ZnCl₂ as a complexantand in part dehydrating agent is consistent with the philosophy of“forcing” the reaction mechanism and this necessarily implies the use ofan anhydrous and controlled reaction environment.

In view of its properties, anhydrous ZnCl₂ was considered particularlysuitable to the desired development of the reaction mechanism. It showedhowever also manageability and treatment difficulties in operation notcompatible with the scale-up of IPA synthesis according to the “soft”philosophy of the present invention.

It was found that using of Zn acetate bihydrate results in a substantialimprovement of operation, yield and quality of the IPA precipitate. Thisimprovement is shown both by the bihydrate and the anhydrous form of theacetate salt. This permits the use of solvents not necessarilyanhydrous, provided that they have the minimum water content.

In addition, zinc acetate bihydrate can be found on the market under adosable non-deliquescent crystalline form which makes a stechiometriccontrol of the reaction easier. It shows practically no solubilizationheat in methanol, this also being in favour of the reaction control.

It is to emphasize that it is a surprising and unobvious issue of thepresent invention the rendering possible the use of a hydrate salt tomake an intermediate product soluble, while the prior art teachingsupposed that the salt had to be anhydrous for a successful result ofthe process. In addition it is to note that this is obtained withoutchanging the stechiometry of the dehydrating base and even substitutingit with a “weaker” one (TEA).

C) Use of picolyl aldehyde (PCA) in lieu of isonicotin aldehyde (ISNA).

Advantages: improved stability on time; improved purity (PCA>99%, ISNAmax 97%); improved reaction and timing control; much higher IPArecovery; much improved IPA quality.

The above cited U.S. Pat. No. 4,808,728 uses ISNA for the reaction ofthe amine function of tryptophan, the Shiff's base formation, therearrangement thereof from aldoimine to ketoimine. This was madepossible by ISNA having the carbonyl-pyridine nitrogen substituents in a1,4- (or para) position.

PCA is a pyridine substituted in orto position (or 2-position) with acarbonyl group of aldehyde type.

By using PCA in lieu of ISNA, on a theoretical basis, it should beexpected an even more “forced” reaction condition and more unstableintermediate products of the reaction. In fact it is neither spontaneousnor easy to attain a rearrangement process of the aromatic structurewhich would result in the formation of a structure of quinoid-1,2 type.At a first sight PCA should not appear a good substitute for ISNA. Thisopinion would be also confirmed by the higher instability of ISNA incomparison to PCA, so that ISNA should appear more reactive than PCA.

Contrary to the above, according to the present invention, PCA resultssuperior to ISNA in view of the stability of the various reactionintermediates and the relative stability thereof. In fact, thestabilization by formation of a Zn complex (in particular with Znacetate which turns out to be more effective than chloride) resulted ina more efficient and organized process intermediate (in favor of energy)with a mechanism of formation completely different from ISNA.

Briefly, this aspect of the present invention makes possible to controlthe course of the reaction by minimizing any interfering products andthus obtaining the highest of yields and definitely improving thequality of IPA.

D) Use of non-anhydrous solvents.

Advantages: Cost abatement; improved control of reaction (no need ofmaintaining anhydrous conditions); simplified procedures.

While the use of anhydrous solvents is compulsory in the methoddisclosed in U.S. Pat. No. 4,808,728, the use of non-anhydrous solventsin the present invention is a logical consequence of the discussionabove. This a further confirmation of the “soft” philosophy at the basisof the present invention, in contrast to the method described in thecited prior art document.

In more recent years, 3-indolylpyruvic acid was the subject of severalpharmacological and clinical studies, as well as of some inventionpatents, in order to clarify its potential uses in experimental studieson animals and in human therapy. E.g., U.S. Pat. No. 4,551,471 describesa process for the biosynthesis of 3-indolylpyruvic acid via tryptophantransamination reaction, and an increase in serotonin levels in thebrain, along with evident pharmacological effects, when the ketoacid wasadministered to rats. U.S. Pat. No. 5,002,963 describes a new method ofbiosynthesis of 3-indolylpyruvic acid, based on the use of L-aminoacidoxydase on tryptophan, as well as the remarkable capability exhibited bythe ketoacid to directly transform in kynurenic acid, and therefore toantagonize the toxic and the degenerative effects due to excitatoryamino acids. U.S. Pat. No. 5,075,329 illustrates the powerfulantiradical properties of 3-indolylpyruvic acid, as well as itspotential pharmacologic applications in cardiopathies, inflammations,arteriosclerosis, etc. U.S. Pat. No. 5,091,172 describes theantioxidizing and antimutagenic properties of 3-indolylpyruvic acid, andits potential use in cosmetics, for protecting skin from oxygen, sun andaging. U.S. Pat. No. 5,210,215 illustrates a new method of chemicalsynthesis of 3-indolylpyruvic acid and of its derivatives, useful indegenerative diseases, based on the use, as starting compounds, ofbenzenes or indoles substituted in the benzene ring. U.S. Pat. No.5,447,951 describes the 3-indolylpyruvic acid capabilities of acting asantistress agent, reducing corticosterone levels in rat.

Concerning pharmacology studies, Bacciottini et al. (Pharmacol. Res.Commun. 19, 803-17, 1987) described 3-indolylpyruvic acid to increasecerebral serotonin turnover in rat, and to possess sedative andanalgesic activities. Russi et al. (Biochem. Pharmacol. 38, 2405-9,1989) demonstrated a marked increment of kynurenic acid in all tissuesof rats, following administration of 3-indolylpyruvic acid, suggesting anew metabolic pathway for the formation of this important inhibitor ofthe excitatory amino acids. Squadrito et al. (J. Cardiovasc. Pharmacol.15, 102-8, 1990) described the anti-hypertension effects of3-indolylpyruvic acid in different rat hypertension models, relatingthem to the increase in the brain concentrations of serotonin. MerloPich et al. (Acta Physiol. Scand. 139, 583-9, 1990) described theeffects of the ketoacid on sleep and on food ingestion in rat,demonstrating that while the anorexigenic effects disappear after a fewdays of treatment, the sleep enhancing effects are maintained even forchronic treatments. Ferretti et al. (Eur. J. Pharmacol. 187, 345-56,1990) described the effects of 3-indolylpyruvic acid as stimulator ofmelatonin production in rat pineal gland. Bruno et al. (Neurosci. Res.Commun. 8, 137-46, 1991) demonstrated that 3-indolylpyruvic acid and itsderivatives can act as antagonists both on the receptor of the kainicacid and on the NMDA ones, attenuating stimulation of calcium uptake incerebellar neuron cultures. Squadrito et al. (Neurosci. Res. Commun. 9,27-36, 1991) described the reduction effects of 3-indolylpyruvic acid onfood assumption by genetically obese rats (Zucker). Biagini et al.(Biol. Psychiatry 33, 712-9, 1993) analyzed the effects of3-indolylpyruvic acid on corticosterone plasma levels and on theglucocorticoid hippocampal receptors, when the rats were subjected to aset of stressor events. Zoli et al. (Neurochem. Int. 23, 139-48, 1993)reported the effects of 3-indolylpyruvic acid in an experimentalischemia model in rat. Lapin and Politi reported the activities of3-indolylpyruvic acid in mouse, both as anxiolytic agent (Pharmacol.Res. 28, 129-34, 1993) and as inhibitor of the toxic effect of ethanol(Alcohol & alcoholism 29, 265-8, 1994).

Concerning clinical studies, Silvestri et al. (J. Intl. Med. Res. 19,403-9, 1991) reported that six healthy volunteers, treated for somenights with doses of 3-indolylpyruvic acid ranging from 100 to 200 mg,exhibited improvements in sleep similar to those observed withtryptophan or with melatonin. Shamsi et al. (Human Psychopharmacol. 11,235-9, 1996) investigated the effects of 3 doses of 3-indolylpyruvicacid (100, 200 and 300 mg) in 10 young volunteers having sleep problems,reporting that ketoacid has a bland action on sleep, probably mirroringa more marked intervention on stress attenuation.

It is important to underline that, in the works published in the '50sand '60s, the researchers interested in 3-indolylpyruvic acid appeareddivided by several contrasts and controversies, since results obtainedby one team were seldom confirmed by others: this was due above all tothe different methods used to synthesize the ketoacid, as even smallamounts of impurities could lead to rapid deterioration of thesubstance, and to fundamental differences in its behavior in a solution.It is also important to underline that, whilst all involved researcherswere aware of the presence of the tautomers when 3-indolylpyruvic acidwas solubilized in aqueous solutions, in all the works reported inliterature no reference is ever made to specific precautions to beadopted in the ketoacid solubilization. This is due to the fact that,owing to the supposedly easy interconversion, an equilibrium between thetwo tautomers is soon reached, at last determining the physiological andpharmacological end effects.

SUMMARY OF THE INVENTION

Object of the present invention is the enol tautomeric form of3-indolylpyruvic acid and derivatives thereof in a state of chemical andshelf life stability, in a semicrystalline solid state and of an enolpurity not lower than 99% referred to the total acid.

A further object of the invention is the enol form of IPA which furtherconsists exclusively of the (Z) form of its geometric isomers formedaround the double bond.

An additional object of the present invention is the use of the aboverecited forms of IPA for therapeutic use at the level of central nervoussystem, cardiovascular pathologies, and treatment of neuron cells andtumors.

Still an object of the invention is a process for the production of theenol tautomeric form of 3-indolyl-pyruvic acid, comprising reactingtryptophan and an aromatic aldehyde, in the presence of a complexantagent and a proton acceptor amine base, in a reaction solvent andhydrolyzing the product of said reaction in a hot acidic hydrolysis stepat a temperature of 45 to 65° comprising: reacting2-pyridine-carboxyaldehyde alone or in a mixture with triethylamine withthe amine function of said tryptophan, for a condensation reactiontherewith, in the presence of triethylamine as a proton acceptornon-nucleophilic amine base, a complexant agent selected from the groupconsisting of zinc acetate or zinc sulfate, and in an organic polarsolvent as a reaction solvent, selected in the group consisting oftetrahydrofuran, dimethyl-formamide, acetonitrile, and methanol.

Object of the invention are also derivatives of the enolic tautomer ofIPA in the form of alkaline or earth-alkaline salts thereof,therapeutical uses thereof which substantially are the same as that ofenol IPA and a process for the production thereof comprising the step ofneutralizing said enol tautomer of 3-indoly-pyruvic acid with a neutralone of said alkaline or earth-alkaline metals, in a first aprotic polarsolvent, together with a second protic polar solvent as a metalactivator.

An additional object of the invention is the use of the above mentionedcompounds for the pharmaceutical industry, in that for the first timethose compounds are in a form which can be employed in the industry inview of their reliable composition, manageable semicristalline structureand shelf life stability.

As an enol IPA derivative is also an object of the invention the indolyllactic acid which is in an interconversion equilibrium with the claimedenol IPA componds, so that IPA can behave as a precursor of lactic acidor viceversa.

Pharmaceutical compositions comprising the above mentioned compounds arealso an object of the invention.

In the light of the pharmacological tests carried out with the enoltautomer of the present invention, it has now been found, according toanother aspect of the present invention, that:

-   -   1) The conversion between the two tautomers of 3-indolylpyruvic        acid is only partially reversible, and the keto form,        originating in aqueous solutions from the enol, mainly follows        metabolic pathways autonomous and independent from the latter;    -   2) the enol tautomer form is the form mainly accountable for the        relevant biochemical and pharmacological properties, described        in literature when 3-indolylpyruvic acid was used;    -   3) the enol tautomer form appears particularly stable in        biological tissues, owing to the presence of specific enzymes        (tautomerase), which hinder conversion to the keto form.

Accordingly, a further object of the present invention are theapplications for therapeutic use of the enol in form essentially freefrom keto tautomer, previously ascribed to the tautomer mixture,exploiting the easy dissolving in physiological environments of enol andof its salts, the determination of the enol tautomer concentrations inanimal tissues and plasma, and lastly the biochemical andpharmacological effects attained when the tautomer, or its salts, areadministered to experimental animals or to human cell cultures.

EXPERIMENTAL PART 1. Synthesis of the Enol Tautomer of 3-IndolylpyruvicAcid and of its Salts

A) process for the production of the enol of 3-indolylpyruvic acidthrough a reaction of tryptophan and 2-pyridine carboxy aldehyde in thepresence of a complexing agent and of triethylamine as amine base in apolar organic solvent, and hot hydrolysis of the reaction product.

B) preparation of alkali and alkali-earth metal salts, prepared bydirect or indirect neutralization of the enol of 3-indolylpyruvic acid,yielded by the synthesis described in A).

In accordance with the invention, hereinafter the qualifying data of thesyntheses identified in A) are described.

As aromatic aldehyde, preferably 2-pyridinecarboxaldehyde, also calledpicolinaldehyde or PCA, is used, under conditions of excess with respectto tryptophan, or of slight defect, compensating the lacking amount witha tertiary amine base. Above all the second combination leads to asubstantial increase in the recovery yield of the enol of3-indolylpyruvic acid, also by virtue of an improved control of thereaction temperatures, due to the fact that PCA develops lesssolubilization heat.

As complexing agent various organic and inorganic zinc salts haveoptionally been used, hydrate as well as anhydrous, also dissolved in anorganic solvent, not necessarily the same one of the coupling reaction.An exemplary selection is: zinc sulfate, zinc acetate (anhydrous as wellas hydrate) and zinc chloride dissolved in anhydrous tetrahydrofuran. Inparticular, the best recovery yields are attained using hydrate zincacetate, previously vacuum stored on a drying agent such as, e.g.,potassium hydroxide (KOH) pellets. Moreover, the hydrate zinc acetate,thus stored, forms, with the intermediates of the coupling reaction,complexes that are more soluble than those of the other salts.

As proton acceptor and dehydrating tertiary amine base triethylamine waspreferentially used, forming intermediate salts more soluble withrespect to the corresponding ones of other known bases having similarfeatures, like DBU or other trialkyl alicyclic bases (DIEA and thelike). Moreover, with respect to the indicated bases, triethylaminedevelops less solubilization heat, allowing an improved control of therun of the reaction temperature, above all at the critical step ofdehydrating, and the entailed structural rearrangement of the couplingintermediate.

As reaction solvent polar solvents are used, not necessarily anhydrous,like DMF, acetonitrile, THF or alcoholic solvents having a shortaliphatic chain, because of the improved solubilization and solvation ofthe reaction intermediates and the maximum solubility at the end stageof high-temperature acid hydrolisis. In particular, the bestperformances are attained using methanol.

The reaction may be conducted at room temperature or, preferably, at lowtemperature, at which the best results are obtained, according to thespecific combination of reactants used. To improve the quality of theenol precipitate of 3-indolylpyruvic acid, the acid hydrolisis processshould be conducted under hot conditions at 45 to 65° C., preferably at55° C. The acid environment should be for a mineral acid, preferably 1Nhydrochloric acid, diluted with a suitable amount of distilled water.

Overall, with respect to the preparations of 3-indolylpyruvic aciddescribed in the above cited patent, the process for the preparation ofthe enol of 3-indolylpyruvic acid object of the invention entails asubstantial improvement of the end recovery yields (>80%), a higherpurity in enol form (>99.9%), an improved quality of the features of theprecipitate (pale yellow color, near-crystal consistence) and theconsequent improvement in the pharmaceutical processability of thesolid. Moreover, these features make this precipitate particularlysuitable for the subsequent preparations, described in the invention, ofalkali and alkali-earth metal salts. The latter were prepared with thedual purpose of making the enol form of 3-indolylpyruvic acidimmediately soluble in saline and of detecting potential salt-specificpharmacological activities.

In accordance with the invention, hereinafter the qualifying data of thesyntheses, identified at B), are described.

The preparation of the salts of the enol of 3-indolylpyruvic acidinvolved alkali and alkali-earth metal salts, and in particular those ofSodium, Lithium, Potassium, Magnesium and Calcium.

The salts were prepared by neutralization of the enol of3-indolylpyruvic acid and of a suitable chemical form of the alkali andalkali-earth metals described in the invention, in a suitable anhydrousnon-protic polar solvent, in particular diethylic ether ortetrahydrofuran, in an amount such as to ensure both the completesolubilization of the indole alpha-ketoacid and the completeprecipitation of the generated salt.

In particular, the alkali and alkali-earth metals were used as neutralmetal (when possible) together with a set amount of an anhydrous proticpolar solvent, specifically methyl alcohol or ethyl alcohol, as metalactivator.

In particular, the alkali and alkali-earth metals were used in thechemical form of the corresponding methoxides or ethoxides, whenpossible, using a known-titre alcoholic solution in an alcoxide andcalculating the adequate amount of alcoholic solution required for theexact neutralization of the enol of 3-indolylpyruvic acid, in an amountby volume of non-protic solvent such as to ensure the quantitativeprecipitation of the generated salt.

Report on structural analysis of indolyl 3-pyruvic acid (IPA) carriedout through NMR spectroscopy.

The IPA structure has been determined by means of NMR spectroscopy, byperforming proton (1-H: 200 MHz) and Carbon 13 (13-C: 50,3 MHz) spectrain deuterated tetrahydrofuran (THF-d8).

All NMR signals related to the nine hydrogen and eleven carbon atoms ofthe molecule have been detected and allotted to the specific positionsof the IPA molecule, which is present in THF-d8 (as well as indeuterated methanol) as 100% enolic tautomer, in the unique (Z) typeconfiguration.

1) - First set of spectra.

A set of proton spectra have been performed at a 0,5 mg/ml concentrationin THF-d8. In these conditions all the signals relative to the ninehydrogens (protons) of the molecule can be clearly observed.

In particular the following allotments can be assigned: COOH 11.25 ppmNH 10.45 ppm OH  7.75 ppm (doublet by H₁₀,OH - J = 1.8 Hz coupling H₁₀ 6.85 ppm (doublet by H₁₀,OH - J = 1.8 Hz coupling) H₂  7.93 ppm(doublet by H₂,NH - J = 2.7 Hz coupling) H₄  7.64 ppm (with only oneorto coupling) H₇  7.32 ppm (with only one orto coupling) H₆  7.12 (withtwo orto couplings) H₅  7.02 (with two orto couplings)

The allotment of the positions relative to the pair of signals H₄ and H₇and to the pair of signals H₅ and H₆ is based on the analogy with thespectrum of indole itself (P. J. Block, M. L. Heffernan, Austr. J. Chem.(1965), 18, 353). The high coupling constant H₁₀,OH suggests that H₁₀ isin trans to OH (Z configuration). Additional spectra evidences are shownin paragraph 7 of this report. On the whole the spectra are consistentonly with the enolic tautomer with 100% purity.

2) Second set of spectra

To the solution of the first set above DO₂ (deuterated water) was added.A disappearance of the COOH and OH proton signals due to the isotopicsubstitution and at a same time the appearance of the H₂O signal isobserved. Moreover, the disappearance of the H₁₀,OH constant confirmsthe previous allotment. As the NH proton signal is less mobile, it doesnot disappear by deuterium exchange quickly.

3) Third spectra set.

The same sample as the second set, but recorded some days later, isused. The disappearance also of the NH proton signal is observed, aswell as the resulting disappearance of the H₂,NH coupling. Furthermorewhile the signals allotted to protons H₅, H₆, H₇ do not change withrespect to the second set, the ones allotted to H₄ become narrower, thusshowing the disappearance of a small coupling constant (about 0,8 Hz)which is known to be present in indoles.

4) Fourth spectra set

Proton spectra in THF-d8 with concentrated IPA solution.

These are substantially similar to the first set with some shiftdisplacement (particularly the COOH group) due to the differentconcentration. Right in view of this the OH mobile protons exchange morequickly with each other and with the mobile protons of COOH causing awidening of the corresponding signals and again the disappearance of thecoupling constant H₁₀,OH.

5) Fifth spectra set

13-C spectra of the solution prepared for the fourth set.

The eleven signals corresponding to the eleven carbon atoms areobserved. The signals of lower intensity (five signals) are associatedto the quaternary carbons and can be so allotted: COOH: 169.0 ppm C₈138.5 ppm C₉ 129.5 ppm C₃ 112.9 ppm

The signals allotted to C₃, C₈ and C₉ have been identified by analogywith the indole spectrum (C. Levy, R. Lichter, G. L. Nelson “NMRSpectroscopy” 2^(nd) Edition, Academic Press (1980), page 1213). The C₁₁allotment to 140 ppm is attributed by exclusion and fulfills theallotment of all five quaternary carbon atoms.

Again by analogy to the indole spectrum the signals of the following CHcan be allotted:

C₄, C₅ and C₆: range 123,3-121,6-120,2 ppm,

C₇: 113,4 ppm.

The signals of the four carbon atoms C₄, C₅ and C₆ are too near to eachother, both in indole and the IPA itself, to allow an individualallotment. Moreover the presence of the substituent in 3-position of IPAcan modify the trend and invert some allotments in comparison to indole.For a reliable allotment the spectra described in the following set havebeen performed.

6) Sixth spectra set

The same solution as employed in the fifth set was used to record abidimensional spectrum which correlates the proton spectrum, through theCarbon-Hydrogen direct coupling factor, to the 13-C spectra (HETCORspectra).

Hence, by knowing the proton spectrum, it has been possible to fulfilthe CH allotment as follows: 105.7 ppm C₁₀ (it correlates with H₁₀ at6.9 ppm) 129.8 ppm C₂ (it correlates with H₂ at 7.9 ppm) 120.25 ppm  C₄(it correlates with H₄ at 7.8 ppm) 121.6 ppm C₅ (it correlates with H₅at 7.02 ppm) 123.9 ppm C₆ (it correlates with H₆ at 7.12 ppm)

In addition it is confirmed the correlation of C₇(113,4 ppm) to H₇ (7,35ppm).

Thus the sequence of all the carbon atoms is not ambiguously allotted.

7) Seventh spectra set

This set shows the carbon spectra fully coupled with the varioushydrogen atoms, in comparison to the normal uncoupled spectra (see thefifth set).

-   -   a) Six carbons become doublets (with a further fine structure)        with coupling constants C-H>=150 Hz, confirming that they are        sp²-hybridated CH. The other five carbon atoms, which are        quaternary carbons, have only very small couplings.    -   b) The COOH signal at 169 ppm is a doublet (J=3,7 Hz) due to the        coupling with H₁₀. It is known that in substitued ethylenes the        carbon-hydrogen couplings separated by three bonds (type        J_(CCCH) factors) are in the order of 3-8 Hz if they are in a        cis configuration and 8-14 if in a trans configuration. When        electronegative groups are present, as in the case of IPA, the        values of the coupling factor correspond to the lower limits of        the indicated ranges (A. W. Douglas, Org. Magn. Reson.; (1977)        9, 69; H. Vogali, W. V. Philipsborn, Org. Magn. Resn.;(1975) 7,        617). As an example in compound CH₃(AcO)C═CH₂, which has an        oxygen linked to the double bond as in the case of IPA, the        coupling of the methyl-carbon and the ethyl-hydrogen is 3,4 Hz        in-the cis-configuration, while it is 8,1 Hz in the        trans-configuration. The detection of a J_(CCCH) equal to 3,7 Hz        confirms for certain that H₁₀ and COOH are in cis-configuration        to one another. Hence the IPA configuration is of (Z)-type, as        additionally already suggested by the coupling constant        J_(H10,OH) being 1,8 Hz in the proton spectra of the first set.        In the opposite configuration (E) the coupling constant J_(CCCH)        would have had a value of about 8 Hz.    -   c) The signal at 140 ppm is a doublet (J=2 Hz) of all of both        ethylene and aromatic quaternary carbons of IPA. Only C₁₁ can        have a single coupling,. particularly with H₁₀. Consequently,        results confirmed the allotment of the 140 Hz signal to C₁₁,        made by exclusion in the discussion of the fifth set of spectra.    -   d) The remaining quaternary signals (C₃, C₈ and C₉) show a        plurality consistent with the above results    -   e) The 129,8 ppm signal shows a direct coupling constant J_(CH)        of 187,5 Hz, far larger than all other CH's, whose values range        from 157 to 160 Hz, typical of sp² carbons. Hence the 129,8 ppm        carbon (differently of the other CH's) should be linked to an        electronegative atom, such as the indole nitrogen, thus        confirming the previous allotment of C₂.

Exemplary preparations of the process according to the invention (A) arereported hereinafter:

EXAMPLE 1 Preparation of the Enol Tautomer Form of 3-Indolylpyruvic Acid

8.0 g tryptophan and 6.0 ml triethylamine are quickly added to 56 mlmethanol, maintained at ice bath temperature. The whitish suspension isleft under stirring, checking the temperature.

After about 10 min, 5.59. ml picolilaldehyde (PCA,2-pyridinecarboxaldehyde) are added, quickly and under stirring,obtaining a clear canary yellow solution. Ice bath temperature andstirring are maintained for other 30 min.

Then, 5.166 gr zinc acetate dihydrate, stored overnight under vacuum andKOH pellets were quickly added, obtaining, after some minutes, a veryfine and fluid canary yellow precipitate.

After 30 min, 12.0 ml triethylamine are added, maintaining it in the icebath and leaving the yellow suspension under stirring for an additional30 min.

The suspension is transferred, under vigorous stirring and for about 15min, in 800 ml 1N HCl, preheated at 55° C., accurately washing thereaction flask with additional 20 ml 1N HCl.

A clear yellow-orange solution, and, after about 5 min from the start ofthe transferring, a noticeable fine and near-crystal yellow-pale goldprecipitation increasing with time, are obtained.

After about 30 min from the end of the transferring, and maintaining thestirring, the heating bath is replaced by a circulating cold water bath.

After 30 more minutes, the precipitate is recovered by vacuum filtrationand is washed successively with 30 ml cold acid water (x2) and once withcold distilled water.

The precipitate is dried overnight in a vacuum dryer, recovering 5.370 g(about the 67.4%) of indole-3-pyruvic, as a fine and semi-crystalyellow-pale gold powder, with >99.9% purity (HPLC, NMR, IR and UV inagreement).

EXAMPLE 2 Preparation of the Enol Tautomer Form of 3-Indolylpyruvic Acid

8.0 g tryptophan and 6.0 ml triethylamine are quickly added to 56 mlmethanol, maintained at ice bath temperature. The whitish suspension isleft under stirring, checking the temperature.

After about 10 min, 4.47 ml picolilaldehyde (PCA,2-pyridinecarboxaldehyde) and 2.73 ml triethylamine are added, quickly,successively and under stirring, obtaining a clear canary yellowsolution. Ice bath temperature and stirring are maintained for 30 moreminutes.

Then, 5.166 g zinc acetate dihydrate, stored overnight under vacuum andKOH pellets are quickly added. The solution changes to dark orange, andafter a few minutes, a very fine and fluid canary yellow precipitate isobtained.

After 30 min, 12.0 ml triethylamine are added, maintaining it in the icebath and leaving the yellow suspension under stirring for other 30 min.

The suspension is transferred, under vigorous stirring and in about 15min, into 800 ml 1N HCl, preheated at 55° C., accurately washing thereaction flask with additional 20 ml 1N HCl.

A clear yellow-orange solution is obtained, in which, after about 5 minfrom the start of the transferring, a noticeable fine and near-crystalyellow-pale gold precipitation appears, increasing with time.

After about 30 min from the end of the transferring, and maintaining thestirring, the heating bath is replaced by a circulating cold water bath.

After further 30 min, the precipitate is removed by vacuum filtrationand is washed successively with 30 ml cold acid water (×2) and once withcold distilled water.

The precipitate is dried overnight in a vacuum dryer, recovering 6.500gr (about the 81.7%) of indole-3-pyruvic, as a fine and semi-crystalyellow-pale gold powder, with >99.9% purity (HPLC, NMR, IR and UV inagreement).

Exemplary preparations of the salts according to B) of the invention areillustrated hereinafter.

EXAMPLE 3 Preparation of the Sodium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Sodium Methoxide in Methyl Alcohol

300 mg of 3-indolylpyruvic acid in the enol form are dissolved, understirring and at room temperature, in 10 ml anhydrous methyl alcohol. Atcomplete dissolution, 2.46 ml 0.5M sodium methoxide solution inanhydrous methanol, freshly prepared, and in a quantity such as toneutralize all the acid, are added. After stirring for one hour at roomtemperature, the reaction mixture is concentrated under vacuum to aboutone third, and added dropwise to 200 ml anhydrous ethyl ether undervigorous stirring, obtaining a very fine yellowish precipitate, whilethe solution tends to discolor. After about two hours, the precipitateis recovered on sintered glass filter (G4 type) by suction filtering.The precipitate thus recovered is washed twice with 10 ml anhydrousethyl ether and kept overnight under vacuum on KOH pellets. 272 mg (>98%yield) sodium salt of the enol form of 3-indolylpyruvic acid (>98%purity HPLC in free acid) are recovered.

EXAMPLE 4 Preparation of the Sodium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Sodium Ethoxide in Ethyl Alcohol

With a procedure similar to that of example 3, the sodium salt of theenol form of 3-indolylpyruvic acid was prepared from sodium ethoxide inanhydrous ethyl alcohol (21% w/w, amount used equal to acidneutralization). Amount of enol tautomer form of 3-indolylpyruvic acid,reaction solvent, reaction yield and purity are identical to those ofexample 3.

EXAMPLE 5 Preparation of the Sodium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Sodium Methoxide Powder

300 mg enol form of 3-indolylpyruvic acid are suspended in 70 mlanhydrous ethyl ether and kept under stirring at room temperature untilcomplete dissolution, obtaining a deep yellow-orange solution. 70.45 mgsodium methoxide powder (>95% purity), in an amount such as toneutralize all the acid, and 963 μl anhydrous methyl alcohol as reactioninitiator are quickly added to this solution, maintaining a vigorousstirring. An abundant fine yellowish precipitation that increases withtime is obtained. The reaction mixture is left overnight under stirringat room temperature. The yellowish precipitate thus obtained isrecovered on sintered glass filter (G4 type) by suction filtering. Theprecipitate thus recovered is washed twice with 10 ml anhydrous ethylether and kept overnight under vacuum on KOH. 262 mg (>98% yield) sodiumsalt of the enol form of 3-indolylpyruvic acid (>98% purity HPLC in freeacid) are recovered.

EXAMPLE 6 Preparation of the Sodium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Metal Sodium

300 mg enol form of 3-indolylpyruvic acid were suspended in 70 mlanhydrous ethyl ether and kept under stirring and at room temperatureuntil complete dissolution, obtaining a deep yellow-orange solution. Tothis solution, 34 mg metal sodium cleaned, quickly weighted and in anamount such as to neutralize all the acid, and 500 μl anhydrous methylalcohol as metal sodium activator were added. A flocculent yellowprecipitation that increases with time is obtained. It is left overnightunder stirring, and the precipitate is recovered as in example 5.251 mg(>93% yield) sodium salt of the enol form of 3-indolylpyruvic acid (>98%purity HPLC in free acid) are recovered.

EXAMPLE 7

With a procedure similar to that of example 5, the following wereprepared:

-   -   potassium salt of the enol tautomer of 3-indolylpyruvic acid        starting from the potassium methoxide powder;    -   lithium salt of the enol tautomer of 3-indolylpyruvic acid        starting from the lithium methoxide powder.

Recovery yields and purities of the corresponding salts are similar tothose of example 5.

EXAMPLE 8

With a procedure similar to that of example 3, the lithium salt of theenol tautomer of 3-indolylpyruvic acid was prepared starting fromlithium methoxide in anhydrous methanol. Recovery yields and purities ofthe corresponding lithium salt are similar to those of example 3.

EXAMPLE 9

With a procedure similar to that of example 6, the potassium salt of theenol tautomer of 3-indolylpyruvic acid was prepared, starting from themetal potassium activated in situ with anhydrous methyl alcohol.Recovery yields and purities of the corresponding potassium salt aresimilar to those of example 6.

EXAMPLE 10 Preparation of the Magnesium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Metal Magnesium

300 mg enol form of 3-indolylpyruvic acid are suspended in 70 mlanhydrous ethyl ether and kept under stirring at room temperature untilcomplete dissolution, obtaining a deep yellow-orange solution. To thissolution 17.9 mg metal magnesium, cleaned, quickly weighted and in aquantity such as to neutralize all the acid, and 500 μL anhydrous methylalcohol as metal sodium activator are added. There ensues a flocculentyellow precipitation increasing with time. It is left overnight understirring, and the precipitate is recovered as in example 5. 300 mg (>95%yield) magnesium salt of the enol form of 3-indolylpyruvic acid (>98%purity HPLC in free acid) are recovered.

EXAMPLE 11 Preparation of the Magnesium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Magnesium Methoxide

300 mg of the enol form of 3-indolylpyruvic acid were suspended in 70 mlanhydrous ethyl ether and kept under stirring at room temperature untilcomplete dissolution, obtaining a deep yellow-orange solution. To thissolution, maintaining a vigorous stirring, 1.18 ml magnesium methoxidein anhydrous methyl alcohol solution (6% w/w concentration) were quicklyadded, in an amount such as to neutralize all the acid. An abundant fineyellowish precipitation that increases with time is obtained. Thereaction mixture is left for three hours under stirring and at roomtemperature. The very fine yellowish precipitate thus obtained isrecovered by filtration on a paper filter (Wathman No. 42, washed withanhydrous ethyl ether and brought to weight constancy) under lightsuction. The precipitate, thus recovered on paper filter, is washedtwice with 10 ml anhydrous ethyl ether and kept overnight under vacuumon KOH pellets. 282 mg (>98% yield) magnesium salt of the enol form of3-indolylpyruvic acid (>98% purity HPLC in free acid) are recovered.

EXAMPLE 12 Preparation of the Calcium Salt of the Enol Tautomer of3-Indolylpyruvic Acid from Calcium Methoxide Powder

With a procedure similar to that of example 5, the calcium salt of theenol form of 3-indolylpyruvic acid was prepared, starting from calciummethoxide powder. Recovery yields and purity of the correspondingcalcium salt are similar to those of example 11.

2. Identification of the enol form of 3-indolylpyruvic acid in solution

The enol form of 3-indolylpyruvic acid, obtained at a >99.9% purity withthe method reported above in examples 1 and 2, appears wholly insolublein water and in chloroform, slightly soluble in ethyl ether, and highlysoluble in acetone, methanol and ethanol. The solution in methanolexhibits, under UV light, the characteristic peak at 325 nm, allowing todistinguish the tautomer from the keto form, in which the peakdisappears and instead the 280 nm peak of the indole ring predominates.The enol form of 3-indolylpyruvic acid appears extremely stable inmethanol, and even after several days at room temperature no relevantlosses are detected.

When the enol tautomer of 3-indolylpyruvic acid is solubilized in anaqueous environment, the decline of the UV peak at 325 nm, more or lessquick according to the solution temperature, the pH and the type ofbuffer used is immediately observed. In the light of the publishedworks, this demonstrates the establishing of the well-known keto-enolequilibrium, which in aqueous solutions largely favors the keto tautomerwhen the aqueous solutions have a neutral or basic pH. In theexperimental conditions used, a quicker disappearance of the 325 nm peakis observed when the enol tautomer of the ketoacid is solubilized indiluted NaOH solutions; it follows that in this solvent the keto-enolequilibrium shifts towards the keto form in a few seconds.

However, contrarily to what described in the previous literature, theketo form in the aqueous solutions is no more than minimally capable ofreconstituting the starting enol form (characterized by the 325 nm peakin UV), unless extremely drastic conditions are used, such as theaddition of concentrated acid solutions; in this case as well, the 325nm peak appears extremely unstable. Therefore, the studies carried outwith the near-pure form of the enol tautomer indicate that in alkali orneutral aqueous solutions rather than an actual keto-enol equilibriumthere occurs a more or less quick transformation of the enol form in theketo form, without appreciable keto-to-enol reversion.

More accurate studies, obtained eluting the solutions in high pressureliquid chromatography (HPLC with Bondapack C18 column) and revealing theindoles, allowed to confirm the more or less rapid disappearance of theenol form in the neutral or basic aqueous solutions, accompanied by theincrease, already in short times, of a new chromatographic peakcharacterized by the keto form of its gem-diole and not by that of theketoacid. At longer incubation times also the gem-diole form disappears,without giving rise to the keto form.

In particular, using a phosphate solution at pH 7.4, it is observed thatthe enol form drops to the 44% already at +30 min, whereas the gem-dioleform begins to predominate (increase) at +1 hour (44% vs 23%), yet itstabilizes at about +2 hours (49%), and then gradually declines.

In short, the tests on the keto-enol equilibrium of the near-pure formof the enol tautomer of 3-indolylpyruvic acid lead to conclude that theenol tautomer, upon having been solubilized in neutral or alkali aqueoussolutions, may not anymore be reconstituted in relevant concentrationsfrom the keto tautomer form, and that the keto tautomer is quicklyshifted towards the gem-diole form, which appears unstable andprogresses to further chemical transformations. This entails importantpractical consequences, as the 3-indolylpyruvic acid will exhibit theproperties of the enol form or those of the gem-diole form, depending onthe solubilization conditions used. Moreover, once the equilibrium hasbeen shifted towards the gem-diole form, the chemico-physical,physiological and pharmacological properties of the enol form willeventually be lost.

3. Identification of the enol tautomer form of 3-indolylpyruvic acid insalines

The near-pure enol tautomer of 3-indolylpyruvic acid, obtained by themethod described above in examples 1 and 2, was investigated on rattissue homogenates, in order to highlight its participation in themethabolic pathways characterizing the use of tryptophan and of itsderivatives in mammalian cells. In particular, since the experiments inaqueous solutions (see 2) ) demonstrated that the enol form of theketoacid quickly disappears at neutral or alkaline pH, the behavior ofthe enol form (obtained dissolving the ketoacid, obtained as describedin examples 1 and 2, immediately prior to the experiments) was analyzedin comparison to the keto form (obtained dissolving the ketoacidpurchased from commercial sources in diluted NaOH) in the two mainpathways accessible to the 3-indolylpyruvic acid in mammalian cells: thetransformation into Tryptophan (catalyzed by different transaminases)and that into 3-indolyl-lactic acid, catalyzed by specificdehydrogenases. Using the keto form no significant transformations totryptophan, nor to 3-indolyl-lactic acid was observed, whereas theresults obtained with the enol form highlighted the extraordinarycapability of this chemical species of acting as precursor to bothindole compounds, and of maintaining a good stability also in slightlyalkaline buffer environments (pH 7.4), without shifts toward thegem-diole form observed in the mere aqueous solutions devoid ofbiological material. This phenomenon, ascribed to the presence inbiological fluids of a specific tautomerase already reported inliterature, allows the enol form to be subjected to biochemicaltransformations by specific enzymes active on the indoles. Inparticular, when the enol form of the ketoacid is incubated in rat liverhomogenates, a gradual transformation of the enol of 3-indolylpyruvicacid in tryptophan is observed: the transformation is rather linear intime, is independent from the pH—at least in the 6.5÷7.5 interval—and itleads to the conversion of over the 50% of the ketoacid in 5 hours. Theonly other indole identified in the reaction is 3-indolealdehyde, whoseconcentration however is modest and does not change with time. Notransformation into 3-indolyl-lactic acid was observed in liverhomogenates.

Conversely, when the enol of 3-indolylpyruvic acid is incubated in ratbrain homogenate, the bulk, of transformation occurs towards the3-indolyl-lactic acid pathway, whereas more modest amounts are convertedto tryptophan, and small amounts are found as 3-indolealdehyde, whoseconcentration substantially does not change over time. The conversion inbrain tissue appears quicker, and in two hours about the 50% of theketoacid is found as 3-indolyl-lactic acid.

In short, the experiments carried out with the two tautomer forms of3-indolylpyruvic acid on mammalian organ homogenates allow to state thatthe enol form, unstable in mere aqueous solvents at physiological pH, isstabilized in tissues, by the intervention of one or more tautomerases,and it represents the substrate used by cells both for the pathwayleading to tryptophan and for that leading to indolyl-lactic acid.Moreover, the discovery that the 3-indolyl-lactic acid appears as thepredominant metabolite in brain after incubation of the enol tautomer of3-indolylpyruvic acid suggests that the 3-indolyl-lactic acid actuallyserves as a reservoir, from which the indole alpha-ketoacid may, whenrequired, be obtained, and which may therefore be used in lieu of theketoacid for some of the pharmacological effects carried out thereby onthe Central Nervous System.

4. Absorption of the enol tautomer form of 3-indolylpyruvic acid

Since the experiments on rat organ homogenates highlighted the peculiarcapability by the enol form of 3-indolylpyruvic acid of remainingstable, by tautomerase intervention, over sufficiently long times,studies were carried out for determining ketoacid levels afteradministration to animals via oral route. Due to the acid environment ofthe stomach (known to favor enol form stability) and to the presence oftautomerase in tissues, the enol form of the ketoacid was demonstratedto be well absorbed and to give plasma levels which, exhibiting a peakbetween +1 and +2 hours, are still clearly identifiable after severalhours.

For instance, when the enol form of 3-indolylpyruvic acid was acutelyadministered by gastric lavage to rats at a dose of 100 mg/Kg, at +1hour 74±14 micrograms(mcg)/ml of plasma were found, whereas ratschronically treated with the same dose exhibited plasma levels equal to183±78 mcg/ml. Raising the dose to 400 mg/Kg, values of 160±13 mcg/mlfor acute administration and of 189±68 mcg/ml for chronic administrationwere obtained.

In dogs as well analogous results were obtained, as the administration(in food-mixed pellets) of 100 mg/Kg enol form of 3-indolylpyruvic acidled, at +1 hour, to plasma rates of 106±47 -mcg/ml in acute, and of97±17 mcg/ml in chronic. 285 mg/Kg doses produced, at +1hour,,concentrations of 136±17 mcg/ml in acute, and of 120±8 mcg/ml inchronic.

Hence, these results allow to conclude that the enol form of3-indolylpyruvic acid is well-absorbed at a gastric level, and isstabilized under the conditions predominating in biologicalenvironments, so as to be still detectable in plasma even after severalhours.

5. Kynurenic acid formation

It is already known from literature that the 3-indolylpyruvic acid mayfunction, in suitable methabolic situations, as direct precursor to thekynurenic acid, the most important endogenous inhibitor of theexcitatory amino acids. It has also been described (Advances Exptl. Med.Biol. 294, 515-18, 1991) that the enol form of the ketoacid is much moreeffective than the keto one in capturing the free radicals of Oxygenthat facilitate the conversion to kynurenic acid. Therefore, comparisontests were conducted between the enol form of 3-indolylpyruvic acid,obtained according to the method described above in examples 1 and 2,the keto form (obtained solubilizing a standard preparation of3-indolylpyruvic acid obtained from commercial sources in 0.1M NaOH),and a mixture of the two forms (obtained solubilizing the above standardpreparation in a pH 7.4 phosphate buffer). Thus, it has first of allbeen found that, contrarily to the keto tautomer form, the enol form of3-indolylpyruvic acid has a strong neutralizing power, not merelytowards the superoxide anion, but also towards the hydroxyl ions, whichare generally considered to be the most dangerous in cell degenerationphenomena. Moreover, the enol form is found to be 100 to 1000 times morepowerful than the keto one, both in protecting membrane phospholipidsfrom degradation and in blocking luminol chemiluminescence induced by axanthin/xanthin oxidase mixture. Concerning the formation of kynurenicacid, under the same experimental conditions the enol form of3-indolylpyruvic acid always produced from 20 to 100 times morekynurenic acid than the keto form or the mixture of the two forms. Whenthe enol form of 3-indolylpyruvic acid was injected intravenously (200mg/Kg) in baboons, the enol plasma concentration curve gradually droppedfrom the high initial values, remaining at clearly detectable levels fora few hours. Concomitantly, kynurenic acid began to form, reaching aplasma peak at +90-+180 min from the enol injection, with remarkableinterindividual variations. Kinetic curves showed that that at least 2to 10% of the enol form of 3-indolylpyruvic acid was transformed inkynurenic acid.

The results of these tests confirm that the 3-indolylpyruvic acid inenol form is the main precursor to the kynurenic acid, and that thistransformation in mammalian biological fluids in vivo takes place in aconsistent and continuous manner, so that the enol may actually beconsidered as a reservoir, capable of continuously releasing kynurenicacid in the blood stream for several hours.

6. Pharmacological activities

The enol form of 3-indolylpyruvic acid, obtained with the methods heretodescribed in examples 1 and 2, was subjected to several pharmacologicaltests, in order to define its potential therapeutic activities, takinginto account the capability of the enol form of acting as tryptophanprecursor (as demonstrated in the experiments on mammalian tissuehomogenates), as well as the fact that the compound may increse cerebrallevels of indolyl-lactic acid, and continuously produces relevantamounts of kynurenic acid, reckoned to be a powerful inhibitor ofexcitatory amino acids. The tests, carried out in comparison both to theketo form of 3-indolylpyruvic acid (obtained solubilizing a standardpreparation of 3-indolylpyruvic acid obtained from commercial sources in0.1M NaOH) and to a mixture of the two tautomers (obtained solubilizingthe above standard preparation in a pH 7.4 phosphate buffer), enabled toascertain that the enol tautomer of 3-indolylpyruvic acid, administeredto rats both orally and by IP injection, possesses severalpharmacological properties evidenced both on the central nervous system(e.g. prolongation of sleep, central analgesia, protection fromNMDA-induced convulsions, reduction of toxic effects from opiates,anxiolytic and antidepressant effects, stress inhibition) and on thecardiovasculary system (pressure reduction in hypertensive rats, but notin normotensive rats). The observed effects were clearly superior tothose obtained with the mixture of the two tautomers, whereas the ketotautomer per se exhibited no apparent effects on the consideredparameters.

Concerning the salts of the enol tautomer form of 3-indolylpyruvic acid,obtained according to the methods reported in examples 3 to 12, theywere assayed in two models of human cell cultures, melanoma andneuroblastoma, in order to determine their specific pharmacologicalactivities.

The results obtained are reported hereinafter:

EXAMPLE 13 Effect of the Enol Tautomer of 3-Indolylpyruvic Acid and ofits Salts on Melanoma Cell Invasion Through Matrigel

Matrigel is an extracellular biological matrix extracted from theEnglebreth-Holm-Swarm murine sarcoma, composed of the major componentsof the basement membrane like laminin, entactin, collagen IV andproteoglycans. At room temperature, Matrigel forms a gel that canpromote the growth of primary and continuous cell lines, and moreovercan act as medium (substrate) for the adhesion, the differentiation, themigration and the invasion of tumor cell lines. Due to such features, aMatrigel test is commonly used in order to characterize the activity ofsubstances capable of interfering with the motility of tumor cells andof inflammatory response cells. The test is conducted in wellspartitioned by an 8 micron pore size membrane: Matrigel (12.4micrograms) is uniformly spread on the membrane, whereas achemoattractant (600 microliters), consisting of the supernatant of amurine hembryo cell line (3T3) is inserted in the bottom section of thewell. The cells to be tested are seeded in the top section of the well,whereas the compounds that could interfere with their motility areplaced above as well as below the membrane, so as to prevent theformation of a concentration gradient. At the end of the experiment, thecells that have crossed the membrane are counted.

Human melanoma cells (A2058 line) were cultured in MEM medium, in a 5%CO2 at 37° C. Having reached the desired growth level, the (100.000)cells were seeded in the top section of the well. The enol of3-indolylpyruvic acid and its salts, solubilized in the cell culturemedium were placed in the top and bottom section of the well.

Results: Compound Concentration (μM) Inhibition % 3-IPA ENOL 500 97 ± 23-IPA ENOL 250  77 ± 11 3-IPA ENOL 125 44 ± 5 Na salt 250 78 ± 3 Na salt125 52 ± 1 K salt 250 78 ± 3 K salt 125 51 ± 3 Ca salt 140 82 ± 6 Casalt 70  38 ± 10 Li salt 250 91 ± 5 Li salt 125 48 ± 7 Mg salt 130 66 ±7 Mg salt 65  46 ± 14The values relate to the average of three distinct experiments, ± is theStandard Deviation.

As it is apparent from the above Table, both the enol tautomer of3-indolylpyruvic acid and its salts exhibit relevant inhibitory effectson the motility of human melanoma cells through Matrigel. This effect,connected to the discovery that the antagonists of excitatory aminoacids may slow down tumor growth (Nature Medicine, 7, 1010-1015, 2001),suggests that the enol tautomer, and all the more some of its salts,could be usefully adopted in tumor treatment.

EXAMPLE 14 Effect of the Enol Tautomer of 3-Indolylpyruvic Acid and ofits Derivatives on Neuronal Cell Sprouting

Neurons are cells extraordinarily capable of sprouting elongations(neurites), underlying the complex interconnections of the CentralNervous System, and which are generally viewed as a cell health index:in fact, it is known that the neurons of younger individuals arecharacterized by several and long neurites, whereas the neurons of elderindividuals or of persons affected by Central Nervous Systemdegenerative diseases exhibit cerebral areas in which the neurons havefew neurites, and of limited length. It is also known that neuronaltumor cells in general grow with short and few neurites, though, whensubjected to the action of trophic factors, they can arrest growth andstart developing longer and more numerous neurites.

LAN5 human neuroblastoma cells were seeded in 6-well plates (25.000cells/well) in DMEM (Dulbecco's Modified Eagle's Medium) medium,additioned with 10% -fetal serum and 40 micrograms/ml gentamycin. After24 hours, the enol tautomer of 3-indolylpyruvic acid or its derivatives,at an end concentration of 10 μM was added to the culture medium. Thetreatment was continued for 7 days, with medium replacement and additionof the compound under test every three days. At +7 days the cells werephotopgraphed, using a Leitz Dialux 20 microscope. Then, neurite lengthwas measured.

The results, reported in the following Table, express, in microns, theaverage length of the neurites for each treatment; ± is the standarddeviation: TREATMENT VALUE (micrometers) Untreated 21.8 ± 8.8  3-IPAEnol 31.9 ± 6.8  3-Indolyllactic acid 39.7 ± 11.8 3-Indolylacetic acid23.3 ± 10.5 5-Hydroxy-Indolylacetic acid 25.7 ± 13.2 Enol Na salt 28.3 ±16.4 Enol K salt 44.1 ± 12.1 Enol L salt 29.4 ± 8.5  Enol Mg salt 40.1 ±17.5 Enol Ca salt 32.7 ± 8.3 

The results are of great interest, and show that the enol tautomer of3-indolylpyruvic acid and some of its derivatives are capable ofstimulating the neurons of a human tumor cell line to sprout neurites,which generally are a symptom of differentiation and vitality. Theseeffects may be partly due to the capability exhibited by antagonists ofthe excitatory amino acids of reducing the growth of neuronal tumors(Nature Medicine, 7, 1010-1015, 2001), yet they are mainly due to theremarkable antioxidizing effects shown by the enol tautomer of3-indolylpyruvic acid. It should be stressed that, among the assayedindoles, only the indolelactic acid exhibits noticeable effects onneurite length, even superior to those of the enol form, (probably sinceit acts as reservoir, continually supplying a compound havingantioxidizing activity), whereas the 3-indoleacetic acid and the5-hydroxy-indoleacetic acid, whose antioxidizing power is very limited,do not influence neurite length under the experimental conditions used.

7. Formulations

Due to its instability in aqueous solutions, the enol tautomer of3-indolylpyruvic acid cannot be formulated in pharmaceutical form withthe common water-using technological systems, and specific protectionsystems shall be resorted to. Acceptable forms may be capsules andtablets for oral use (in this latter case with technologicalcontrivances, for the manufacturing as well as for the coating and theoptional presence of absorption retarding agents), and powders to besolubilized immediately prior to use, for injectable products, using anyone of the salts described in the invention.

The dosage envisaged for human use ranges from 100 to 500 mg per dose.

1-36. (canceled)
 37. Enol tautomeric form of 3-indolylpyruvic acid or aderivative thereof consisting of the (Z) form of the geometric isomer ofsaid enol tautomeric form and in a state of a chemically and shelf lifestable semicrystalline solid having an enol purity not lower than 99%referred to the total acid.
 38. Enol tautomeric form of3-indolyl-pyruvic acid or a derivative thereof, according to claim 37,having an enol purity of 99.9%.
 39. A process for the production of theenol tautomeric form of 3-indolylpyruvic acid consisting of the (Z) formof the geometric isomer of said enol tautomeric form and in a state of achemically and shelf life stable semicrystalline solid having an enolpurity not lower than 99% referred to the total acid, comprising:reacting 2-pyridine-carboxyaldehyde alone or in a mixture withtriethylamine with the amine function of said tryptophan, for acondensation reaction therewith, in the presence of triethylamine as aproton acceptor non-nucleophilic amine base, a complexant agent selectedfrom the group consisting of zinc acetate and zinc sulfate, in anorganic polar solvent as a reaction solvent selected in the groupconsisting of tetrahydrofuran, dimethylformamide, acetonitrile, andmethanol, and hydrolyzing the product of said condensation reaction in ahot acidic hydrolysis step at 45 to 65° C.
 40. A process according toclaim 39, wherein said complexant agent is Zn acetate dihydrate, or Znsulfate heptahydrate.
 41. A process according to claim 39 wherein thetemperature of said hot acidic hydrolysis step is 55° C.
 42. A processaccording to claim 39, wherein said reaction solvent is a not anhydroussolvent.
 43. A derivative of the enol tautomeric form of3-indolyl-pyruvic acid according to claim 37, wherein said derivative isa salt with an alkali or earth-alkaline metal, in a state of chemicalpurity, shelf life stability and crystalline form.
 44. A salt derivativeaccording to claim 43, wherein said alkali or earth-alkaline metal issodium, lithium, potassium, magnesium or calcium.
 45. A process for theproduction of an alkali or earth-alkaline metal salt derivative of theenol tautomeric form of 3-indolyl-pyruvic acid, comprising neutralizingsaid enol tautomer of 3-indoly-pyruvic acid with an alkali orearth-alkaline metal, in a first aprotic polar solvent, together with asecond protic polar solvent as a neutral metal activator.
 46. A processaccording to claim 45, wherein said first aprotic and said second proticpolar solvent are anhydrous.
 47. A process according to claim 45,wherein said aprotic polar solvent is diethyl-ether or tetrahydrofuran.48. A process according to claim 45, wherein said neutral metal is ametal methoxide or ethoxide and said protic solvent is methyl- alcoholor ethyl-alcohol.
 49. A process according to claim 45, wherein saidalkali or earth-alkaline metal is sodium, lithium, potassium, magnesiumor calcium.
 50. A method for the manufacture of a medicament in thepharmaceutical industry comprising using the enol tautomeric form of3-indolyl-pyruvic acid or a derivative thereof, as a chemically pure andshelf stable pharmaceutical material in said manufacture.
 51. A methodfor the treatment of disturbances of the central nervous systemcomprising administering a therapeutically effective amount of the enoltautomeric form of 3-indolyl-pyruvic acid or a derivative thereof, to apatient in need thereof.
 52. A method according to claim 51 for thetreatment of at least one of anxiety, depression, sleep disturbances,appetite disturbances, mood and emotionality disturbances, cerebralpost-trauma disturbances, cerebral hypoxia and ischemia, epilepsies,elder cognition declination, neurodegenerative disturbances, physicaland psychological stress situations.
 53. A method for the therapeutictreatment of promoting the sprouting of neuron cells in a patient inneed thereof comprising administering to said patient a therapeuticallyeffective amount of the enol tautomeric form of 3-indolyl-pyruvic acidor a derivative thereof.
 54. A method for the therapeutic treatment of acardiovascular pathologic condition comprising administering to apatient in need thereof a therapeutically effective amount of the enoltautomeric form of 3-indolyl-pyruvic acid or a derivative thereof.
 55. Amethod for the therapeutic treatment of tumors comprising administeringa therapeutically effective amount of the enol tautomeric form of3-indolyl-pyruvic acid or a derivative thereof, to a patient in needthereof.
 56. A method for the therapeutic treatment of a deficiency ofserotoninergic activity or an excess of excitatory amino acid activitycomprising administering to a patient in need thereof a therapeuticallyeffective amount of the enol tautomeric form of 3-indolyl-pyruvic acidor a derivative thereof.
 57. A derivative of the enol tautomeric form of3-indolyl-pyruvic acid according to claim 37, wherein said derivative is3-indolyl lactic acid.
 58. A method for a therapeutic treatment of thecentral nervous system comprising administering to a patient in needthereof a therapeutically effective amount of 3-indolyl-lactic acidaccording to claim 57 as a 3-indolyl pyruvic acid precursor.
 59. Amethod for the therapeutic treatment of a patient in need of3-indolyl-lactic acid administration, comprising administering to saidpatient a therapeutically effective amount of the enol tautomeric formof 3-indolyl-pyruvic acid as a precursor of 3-indolyl-lactic acid.
 60. Amethod for the therapeutic treatment of a patient in need of3-indolyl-lactic acid administration, comprising administering to saidpatient a therapeutically effective amount of an alkaline orearth-alkaline salt of the enol tautomeric form of 3-indolyl-pyruvicacid, as a precursor of 3-indolyl-lactic acid.
 61. A pharmaceuticalcomposition comprising the enol tautomeric form of 3-indolyl-pyruvicacid or a derivative thereof according to claim 37, as well as and apharmaceutically compatible excipient or carrier.
 62. The pharmaceuticalcomposition according to claim 61, wherein said carrier is a physiologicsolution.
 63. The pharmaceutical composition according to claim 61 inthe form of capsules, tablets, suppositories or injectable vials.